| Toxocara canis is a parasitic nematode of Toxocara, Toxocaridae. Various animals, such as canids and foxes, are commonly infected through oral ingestion of embryonated eggs and transplacental transmission of infectious larvae. The infective larvae usually migrate through viscera, and undergo development to adult life-cycle stage in the intestinal tract of definitive hosts, while when they enter into paratenic hosts, in stead of further development, they would migrate to viscera, muscle and/or neural system, and temporarily encyst as arrested infectious larvae. A wide range of animals as well as human beings can be infected by accidental ingestions of infective eggs or infectious larvae in the undercooked meat/viscera. Although there is no obvious syndrome in most infected people, visceral larva migrans(VLM), ocular larva migrans(OLM), and/or covert toxocariasis/common toxocariasis(CT) commonly occur. Importantly, long-term effects of chronic toxocariasis would result in neurological toxocariasis(NT), and public concern has been raised by the important relationship between human toxocariasis and respiratory diseases(e.g. asthma and pulmonary fibrosis), allergic disorders(e.g. pruritus and urticaria) and neurological diseases(e.g. impaired deficits, epilepsy, schizophrenia, dementia and idiopathic Parkinson’s disease). Epidemiological investigations indicated worldwide distribution and high prevalence of Toxocara infection, particularly in tropical and subtropical areas. Unfortunately, there are still difficulties and efficacy problems(sensitivity and specificity) in diagnosis, yet there are no drugs that are effective against arrested, somatic larvae. New diagnostic methods and intervention strategies are desiderated.Phosphorylation and dephosphorylation is the principal mechanism under post transcriptional regulation of protein function. Serine/threonine protein phosphatases(STPs) are the main operator of dephosphorylation in eukaryotes, which can be classified into three families, phosphoprotein phosphatases(PPPs), protein phosphatases magnesium dependent(PPMs) and protein tyrosine phosphatase(PTPs), based on the identities of their amino acid sequences, structure and sensitivity to inhibitors. STPs play roles in a wide range of biological processes including development, reproduction and cellular signal transduction of parasites. Specifically, serine/threonine protein phosphatase 1(PP1) is an important member of PPPs, and plays key roles in regulating various cellular processes, such as cell division, protein synthesis, cytoskeletal reorganization, and signal transduction. Moreover, the roles of PP1 in nucleus positioning, cell proliferation, host-cell attachment, and host-cell invasion have been identified in parasites, suggesting novel drug targets. There is a lack of information about STPs of T. canis.In the current study, the full-length Tc-stp-1 gene was cloned, the tissue distribution and subcellular distribution of Tc PP1cα were investigated, and the biological function of Tc-stp-1 gene was studied by RNA mediated interference assays(RNAi). Results were summarised as follows: 1. Molecular cloning and sequence analysis of Tc-stp-1Rapid amplification of the c DNA ends technology(RACE, 5’) was employed to generate the full-length c DNA of Tc-stp-1 gene. From the 1192 bp sequence, complete coding sequence(CDS) of 942 bp, 5’ untranslated regions(UTR), and 3’ UTR were identified. 312 amino acids sequence were predicted, with the ions binding sites predicted at G61, Y67, D93, T125, L175 and V250, while active sites at G61, Y67, D93, T125, S126, L175 and V250. In addition, multiple alignment analysis indicated that the amino acid sequence predicted from Tc-stp-1 conserved with other sequences at motifs GDXHG, GDXVDRG and GNHE, whereas great differences were found at both ends. Phylogenetic analysis suggested the highest similarities of PP1 catalytic subunits(PP1c) between T. canis and L. loa, B. malayi in terms of amino acid sequences. In addition, based on structure prediction, a Tc PP1cα protein was predicted from Tc-stp-1 gene, with binding sites predicted at D90, R94, N122, H123, R220 and H247, whereas GO annotation inferred singalling transduction(GO:0004871), protein dephosphorlation(GO: 0006470) and cytoplasm(GO: 0005829) in molecular function, biological process and cellular component of Tc PP1cα, respectively.2. Prokaryotic expression of Tc PP1α and polyclonal antiserum preparationThe Tc-stp-1 gene was cloned and used for the construction of Tc-stp-1/p ET 28a(+) expression plasmid, results of sequencing showed that 948 bp fragment(including restriction enzyme recognition sequence) had been inserted. Prokaryotic expression system of Escherichia coli BL 21(DE3) was employed for the expression of recombinant Tc PP1cα. SDS-PAGE analysis showed that the recombinant Tc PP1cα was produced in inclusion bodies at about 30 k D in molecular weight. Expression conditions were optimized to gain considerable protein, under the inducement of 0.4 m M isopropylthiogalactoside(IPTG) and incubation of 4 h for 37 °C. Ni-NTA was used for the purification of target protein, and considerable purified protein was obtained by eluting with 1 M imidazole solution, which was used to produce rabbit anti-Tc PP1cα polyclonal antiserum. The antiserum was characterized as titer > 1:256000, concertration of 0.51 mg/m L, and purity > 95%. By contrast, western blot(WB) analysis indicated high specificity of this rabbit anti-Tc PP1cα polyclonal serum. 3. The specific transcription of Tc-stp-1 and specific tissue distribution of Tc PP1cαQuantitative real-time polymerase chain reaction(q RT-PCR) was utilized to reveal the profiles of Tc-stp-1 in adult female and male worms as well as in the tissues. The quantitative data showed that the Tc-stp-1 was specifically transcribed in male adult T. canis, with high transcriptional level in testis and relatively high level in vas deferens. By contrast, indirect fluorescence immunohistochemical analysis was performed, which indicated that Tc PP1cα expressed exclusively in the male germline tissues, particularly in the testis and vas deferens and germ cells inside. The specific transcription of Tc-stp-1 and distribution of Tc PP1cα in the tissues of male adult T. canis indicated the functional roles of Tc-stp-1 in biological processes during reproduction of T. canis. 4. Subcellular location of Tc PP1αThe coding sequence of Tc-stp-1 gene was amplified for the construction of the recombinant Tc-stp-1/p SL 1180 plasmid, results of sequencing showed that a 948 bp fragment(including restriction enzyme recognition sequence) was inserted into the expression vector. Subsequently, eukaryotic expression system of sf9 cells were employed to produce the recombinant Tc PP1cα. In addition, indirect fluorescence immunocytochemical analysis was carried out, which showed that the Tc PP1cα distributed in cell plasma of inter-mitotic sf9 cells and at the equator of the spindle during dividing stage. The subcellular distribution of Tc PP1cα indicated potential roles in the biological processes of germ cell division of T. canis. 5. RNA interference on Tc-stp-1Double-stranded RNAi(ds RNAi) assays were carried out to interfere the process of transcription-expression of Tc-stp-1. Gene knockdown analysis was performed using q RT-PCR to determine the transcriptional levels of Tc-stp-1. Results showed that the transcriptional level of Tc-stp-1 decreased significantly in testis and vas deferens after 24 h, then decreased in seminal vesicle, rebound in vas deferens after 48 h, and rose in testis, seminal vesicle and vas deferens after 72 h. Microscopy scanning was used to scan the phenotypic aberration. Correspondingly, abnormal morphologies of germ cells were observed in seminal vesicle and vas deferens after 24 h, and blocked meiotic divisions of spermatocytes in vas deferens after 48 h, whereas recovered meiosis in vas deferens after 72 h. Overall, the gene knockdown analysis and relevant phenotypic aberrations indicated essential roles of Tc-stp-1 with regulating kinetochore-microtubule mechanism under spermatogenesis and sperm cell maturation in the male adult T. canis, suggesting a potential drug target. |
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